DESCRIPTION: The goal of this project is to understand the roles of chaperones in the localization of the LamB protein to the outer membrane of E. coli. Protein localization represents a key cellular process. The localization of LamB in the cell envelope entails the secretion of the unfolded LamB polypeptide through the cytoplasmic membrane, the folding and trimerization of LamB in the periplasm, and the insertion of the trimer into the outer membrane in a topologically oriented manner so that the beta barrels which span the cell envelope would form a specific channel for the transport of maltodextrins, and the connecting loops that are extruded in the cell surface would serve as the specific receptor for absorption by phage lambda. LamB has served as a model molecule for studies of protein localization. Although a great deal is known about this protein, the components of the secretion machinery and the initial steps of secretion, our knowledge of the later steps is still sketchy. Of interest is the folding and the assembly pathway which is governed not only by the intra-chain and inter-chain interactions in the protein but also folding enzymes and membrane-associated macromolecules which act as chaperones and ushers. The applicant wants to identify specific chaperone components of the cell envelope and develop an assay for refolding of LamB in which the activity of the chaperones can be tested. The proposal details experiments to pursue three specific lines of study: 1. Study the role of the single disulfide bond in LamB protein folding and assembly, and assess the involvement of DsbA, a disulfide bond forming enzyme located in the periplasm. 2. Test in vitro the ability of known chaperones (a) to catalyze the folding of purified LamB protein, and (b) to prevent aggregation of a "compact" monomer which may represent a metastable folding intermediate. 3. Identify other cell envelope components (proteins, LPS, lipids) which influence the in vitro folding reaction, or whose absence in specific mutant cells affects the in vivo assembly.